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  • DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS (PAHS)

    IN SEAFOOD USING GC-MS: A COLLABORATIVE STUDY

    Lucie Drábová, Jana Pulkrabová, Kateřina Maštovská, Kamila Kalachová, Vladimír Kocourek, Jana Hajšlová

    5th Meeting on Chemistry and Life 2011 14‐16th September 2011, Brno, Czech Republic

  •  AOAC CALL: PAHS IN FISH AND SEAFOOD

     NEW METHOD FOR MULTIPLE POPs

     AOAC: COLLABORATIVE STUDY

    Talk Summary

    5th Meeting on Chemistry and Life 2011

  • Oil Spill - Gulf Of Mexico 2010

    5th Meeting on Chemistry and Life 2011

  • Call for Methods 23/7/2010

    5th Meeting on Chemistry and Life 2011

  • Isolation 10 min

    Clean up 30 min

    Identification & quantification

    1 h

    BFR PCB PAH Non-ortho PCB

    Extraction Shaking (H2O + ethylacetate)

    Partition (transfer into organic phase) induced by MgSO4 + NaCl

    Clean up Silicagel minicolumn

    Identification & quantification GC-QMS(EI)

    GC-TOFMS (EI) GC×GC-TOFMS (EI)

    6 SAMPLES / < 1 HOUR

    SINGLE GC INJECTION

    CONffIDENCE Integrated sample preparation

    5th Meeting on Chemistry and Life 2011

  • Target analytes

    5th Meeting on Chemistry and Life 2011

    Benz[a]anthracene BaA Benzo[b]fluoranthene BbFA Benzo[k]fluoranthene BkFA Benzo[ghi]perylene BghiP Benzo[a]pyrene BaP Chrysene CHR Dibenz[a,h]anthracene DBahA Indeno[1,2,3-cd]pyrene IP Benzo[j]fluoranthene BjFA Benzo[c]fluorene BcFL Cyclopenta[cd]pyrene CPP Dibenzo[a,e]pyrene DBaeP Dibenzo[a,h]pyrene DBahP Dibenzo[a,i]pyrene DBaiP Dibenzo[a,l]pyrene DBalP 5-Methylchrysene 5-MC

    15 + 1 EU PAHs Acenaphthene AC Acenaphthylene ACL Anthracene AN Fluoranthene FA Fluorene FL Naphthalene NA Phenanthrene PHE Pyrene PY

    16 US EPA PAHs

    methylated homologues 2-Methylanthracene 2-MA 1-Methylchrysene 1-MC 3-Methylchrysene 3-MC 1-Methylnaphthalene 1-MN 2-Methylnaphthalene 2-MN 1-Methylphenanthrene 1-MPH 1-Methylpyrene 1-MP 1,7-Dimethylphenanthracene 1,7-DMP 2,6-Dimethylnaphthalene 2,6-DMN

    Benzo[e]pyrene BeP Dibenzothiophene DBT

  • Method Performance Characteristics – PAHs and their homologues (GC-TOFMS)

    750 1000 1250 1500 1750 2000 2250 2500 0

    10000

    20000

    30000

    40000

    50000

    60000

    Time (s) 128 142 152 153 166 178 202 216 226 228 242 252 276 278 302

    Na ph

    1‐M N

    AC L

    AC FL

    DB T

    PH E

    AN

    FA PY

    1‐M PH 2‐M

    A

    Ba A

    CP P

    CH R 

    1‐M C

    5‐M C

    Bb FA

    Bk FA

    BjF A Ba P D

    Ba hA

    IP

    Bg hiP

    DB alP

    DB ae P

    DB aiP

    DB ah

    P

    2‐M N

    3‐M C

    SHRIMPS Recovery: 69 – 109 % Repeatability: 2 – 15 % LOQ: 0.05 – 0.25 µg.kg‐1

    FISH  Recovery: 75 – 107 % Repeatability: 2 – 13 % LOQ: 0.05 – 0.25 µg.kg‐1

    5th Meeting on Chemistry and Life 2011

    DB-EUPAH (20 m × 0.18 mm × 0.14 µm)

  • 21/10/2010

    5th Meeting on Chemistry and Life 2011

  • 5th Meeting on Chemistry and Life 2011

  • Target analytes – AOAC study 1,7-Dimethylphenanthracene 1,7-DMP 1-Methylnaphthalene 1-MN 1-Methylphenanthrene 1-MPH 2,6-Dimethylnaphthalene 2,6-DMN 3-Methylchrysene 3-MC Anthracene AN Benz[a]anthracene BaA Benzo[a]pyrene BaP Benzo[b]fluoranthene BbFA Benzo[ghi]perylene BghiP Benzo[k]fluoranthene BkFA Chrysene CHR Dibenz[a,h]anthracene DBahA Fluoranthene FA Fluorene FL Indeno[1,2,3-cd]pyrene IP Naphthalene NA Phenanthrene PHE Pyrene PY

    5th Meeting on Chemistry and Life 2011

    19 PAHs

  • AOAC Int. Collaborative Study Study phases:

    (1) Laboratory qualification

    (2) Test sample analysis

    Study design:

     3 matrices: mussel, oyster, shrimp  total of 5 different levels of BaP (2 – 50 µg/kg)  other studied PAHs at varying levels from 2 to 250 µg/kg

    that mimic typical PAH patterns  each matrix fortified at 3 different concentration levels in

    duplicate + one blank for each matrix  total of 7 x 3 = 21 study samples

    5th Meeting on Chemistry and Life 2011

  • AOAC Int. Collaborative Study Study participants:  Adpen Laboratories (FL, USA)  Covance Laboratories (WI, USA)  EU PAH Reference Laboratory (Belgium)  Eurofins CAL (LA, USA)  FL Dept. of Agriculture and Consumer Services (FL, USA)  Food Safety Net Services (TX, USA)  Institute of Chemical Technology (Czech Republic)  LECO Corporation (MI, USA)  MD Dept. of Agriculture (MD, USA)  MI Dept. of Community Health (MI, USA)  Microbac Laboratories (IN, USA)  Premier Laboratory (CT, USA)  Silliker JR Laboratories (BC, Canada)  Thermo Fisher Scientific FSRC (Germany)  State Veterinary Institute in Praha, (Czech Republic)

    Study direction team:  Co-study directors: K. Mastovska (Covance), W. Sorenson (Covance), and

    J. Hajslova (ICT)  Technical advisors: J. Schmitz (Covance), J. Pulkrabova (ICT)

    5th Meeting on Chemistry and Life 2011

    15 laboratories

  • (1) Laboratory qualification phase

    Optimization of GC-MS, silica-SPE clean-up and solvent evaporation conditions Check of potential reagent blank contamination Familiarization with the method

    Qualification steps: (1) GC separation test (2) Calibration range test (3) Solvent evaporation test (4) PAH and fat elution profiles (5) Procedure blank test (6) Low-level spike test (7) Practice sample analysis

    5th Meeting on Chemistry and Life 2011

  • 1. GC Separation Test

    (1) a baseline separation of benzo(a)pyrene and benzo(e)pyrene (2) at least 50% valley separation of anthracene and phenanthrene (3) at least 50% valley separation for benzo(b)fluoranthene, benzo(j)fluoranthene, 

    and benzo(k)fluoranthene

    To optimize GC separation of PAHs

    (1) (2) (3)

    BeP

    BaP

    Phe

    AN

    BbFBkFBjF

    Criteria:

    (1) DB‐EUPAH (20m x 0.18 mm x 0.14 µm) (Agilent  J&W, USA) (2) Rxi‐17 Sil (30m x 0.25 mm x 0.25 µm) (Restek, USA) (3) DB‐17 MS (30m x 0.25 mm x 0.25 µm) (Agilent  J&W, USA) (4) TR‐50MS (30m x 0.25 mm x 0.25 µm) (Thermo Fisher Scientific, USA) (5) ZB‐50 (30m x 0.25 mm x 0.25 µm) (Phenomenex, USA)

    GC columns used in the study

    5th Meeting on Chemistry and Life 2011

  • y = 0.0181x + 0.0937 R² = 0.9999

    0 2 4 6 8

    10 12 14 16 18 20

    0 200 400 600 800 1000N or

    m al

    iz ed

    R es

    po ns

    e

    Concentration (µg/L)

    BaP

    y = 0.0215x + 0.1677 R² = 1

    0

    20

    40

    60

    80

    100

    120

    0 1000 2000 3000 4000 5000N or

    m al

    iz ed

    R es

    po ns

    e

    Concentration (µg/L)

    Naph

    2. Calibration Range Test

     8-point calibration curves  Calibration range: 5 –1000 µg/L for BaP and lower levels PAH, 12.5 – 2500 µg/L

    for higher-level PAHs, 25 –5000 µg/L for naphthalene  Coefficients of determination (r2) of 0.990 or greater are required  Test for carry-over (response in the solvent blank < 0.5% of the highest standard)

    To determine linear range for analyte responses normalized to respective 13C-PAHs, test carry-over, and optimize injection conditions

    r2: 0.9987 – 1.000 Max residuals ± 20% 5th Meeting on Chemistry and Life 2011

  • 3. Solvent Evaporation Test To determine absolute recoveries of PAHs and 13C-PAHs during two evaporation experiments simulating the two evaporation steps in the method

     The absolute recoveries - above 70%  Recoveries of the heavier PAHs - in the range of 90-110%

    (a) evaporation of 5 mL of an PAH/13C-PAH solution in EtOAc and reconstitution in isooctane

    (b) evaporation of 10 mL of an PAH/13C-PAH solution in hexane:DCM (3:1, v/v) and reconstitution in isooctane Criteria:

    Evaporation techniques employed in the study:  nitrogen blown-down  rotary vacuum evaporation

    5th Meeting on Chemistry and Life 2011

    Recommendations:  use isooctane as a keeper in both evaporation steps  add 1-2 mL of EtOAc prior to the second evaporation step to improve recoveries of volatile PAHs

    Recoveries : 78 – 109 %

  • 4. PAH and Fat Elution Profiles

    0 10 20 30 40 50 60 70 80 90

    0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 %  A na

    ly te  e lu te d

    Elution volume (mL)

    LIPIDS

    0 10 20 30 40 50 60 70 80 90

    100

    0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

    %  A na

    ly te  e lu te d

    Elution volume (mL)

    Commercial SPE silica cartridge (UCT)

    100 mg of fat loaded on  column containing 1G of

    sorbent

    100 mg of fat loaded on  column containing 1G of

    sorbent

    in‐house  prepared mini ‐

    column

    LIPIDS

    To determine elution profiles of PAHs and fat on the silica gel SPE cartridge of your choice and optimize the elution solvent volume

    5th Meeting on Chemistry and Life 2011

  • 5. Procedure Bl

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